Long-Fragment Nucleic Acid Sequencing by Segmented Read Assembly
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Solution Overview
Problem
Existing sequencing technologies, particularly second-generation sequencing, struggle with high error rates and are inadequate for sequencing long-fragment nucleic acids, which are crucial for studying complex genomic structures like highly heterozygous genomes, highly repetitive sequences, high GC regions, and copy number variations.
Innovation Solution
A method involving segmental sequencing of long-fragment nucleic acids, where adapters are added to both ends of the insert, followed by controlled extension treatments using dNTP mixtures with polymerase reaction blocking and detectable groups to accurately sequence long fragments by splicing multiple segments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If second-generation sequencing technology is used, then throughput is improved and cost is reduced, but read length is limited and error rate increases for long fragments
Solution Approach 1:
The patent divides long-fragment nucleic acid sequencing into multiple segments by using nested primer pairs with different extension lengths. Short primers sequence regions closer to adapters, while long primers sequence regions farther away, allowing accurate sequencing of long fragments (500-2000 bp) by combining multiple read segments without requiring the entire long fragment to be sequenced in a single read.
2Productivity
If second-generation sequencing is used, then high throughput is achieved, but read length capability deteriorates for long-fragment nucleic acids
Solution Approach 1:
The patent segments the sequencing task by designing nested primer pairs where short primers (e.g., 18-22 bp) sequence regions within 100-300 bp of adapters, and long primers (e.g., 30-40 bp) sequence regions 300-800 bp from adapters. This segmentation allows the system to maintain high throughput while effectively sequencing long fragments by combining multiple shorter reads.
Solution Approach 2:
The patent adds a dimensional approach by sequencing from both ends of the insert using forward and reverse primers, creating overlapping reads that cover the entire long fragment. This bidirectional sequencing approach extends the effective read length capability without requiring single-read lengths to exceed the fragment length.
3Productivity
If standard sequencing primers are used without blocking groups, then extension efficiency is high, but specificity deteriorates with multiple primer pairs
Solution Approach 1:
The patent applies local quality by adding polymerase reaction blocking groups (e.g., 3'-O-azide) specifically to dNTPs used in the first extension reaction, while dNTPs in the second extension reaction lack these blocking groups. This localized modification ensures that the first extension is highly specific to the intended primer-template pair, preventing non-specific amplification when multiple primer pairs are used, while maintaining high extension efficiency through optimized polymerase and buffer conditions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables accurate and cost-effective sequencing of long-fragment nucleic acids, overcoming the limitations of high error rates and length constraints in existing technologies, allowing for comprehensive analysis of complex genomic features.
Implementation Method 1
performing a first extension treatment on the nucleic acid molecule to be detected by using a first dNTP mixture and a sequencing primer, the sequencing primer being paired with one of the first sequencing adapter and the second sequencing adapter, and the sequencing primer extending to upstream of the predetermined region
Data Source
AI summary
Provided is a method for sequencing a long-fragment nucleic acid. The nucleic acid molecules each containing a long insert, a first sequencing adapter, and a second sequencing adapter, is used to construct a sequencing library, and the sequencing is performed in segments to sequence the nucleic acids having the long inserts.